沙門氏菌metE基因之表現調控

Abstract

腸道沙門氏菌為世界性之食物媒介疾病，其中Salmonella Typhimurium LT2 菌株之基因組更已確定，與模式細菌大腸桿菌之基因組具高相似性，且對人類之毒性較模式動物小鼠相對為低，可作為研究流行病學病原菌之相對應的模式微生物。也可藉由生理調控機制之研究，以對其更加了解。其中，甲硫胺酸之合成為細菌生存所必需，於合成路徑最後一步之作用酵素MetE 更是合成過程中所必須的重要酵素之一。 因此，於本研究將建構不同之缺失株及相關的系列選殖質體，利用西方墨點法及β-galactosidase 活性分析等不同的基因表現方法，探討革蘭氏陰性菌之沙門氏菌的metE 基因調控情形，並確定其調控是否與核糖開關或小分子RNA FnrS 相關。 結果發現metE mRNA 5’端未轉譯區域的+30~+68 片段缺失會造成metE 的表現量明顯下降，可知metE 5’端未轉譯區域對調控的必要性。並且，metE 之表現量下降差異非受抑制因子MetJ 之影響。而革蘭氏陰性菌腸道沙門氏菌之metE 基因調控機制與革蘭氏陽性菌克勞氏芽孢桿菌的核糖開關之調控機制不同。另外，也發現Hfq 及FnrS 與metE 之表現具有相關性，且小分子RNA FnrS 的大量表現可抑制metE 之表現，與metE mRNA 5’端未轉譯區域可為互補與接合。而兩者任一之結構的缺失皆會降低其對metE 基因表現之影響。 希望透過對此腸道沙門氏菌metE 調控機制之了解，除可更加認識腸道沙門氏菌metE 基因之調控機制外，也期將具可利用性之調控機制應用於其他相類似基因之研究上。

Salmonella enterica is one of the pathogenic bacteria that cause the worldwide food-borne desease. One strain, Salmonella Typhimurium LT2, its genome has been sequenced with a similarity to Escherichia coli. Salmonella enterica is more toxicity in mice than in human, and it can be used for an epidemiological study of human pathogens. However, Salmonella Typhimurium LT2, itself, can be used as a model microorganism for its physiological study. Methionine is an essential amino acid for the bacteria, and in the methionine biosynthesis, the last step was modulated by MetE, which is a key enzyme in this biosynthetic pathway. Here, a set of deletion mutations in metE leader sequences were made; using Western blotting and the assays of β-galactosidase acitivity to analyze metE gene expression. In addition, we also tested whether it is related to the regulation of riboswitch or is controlled by small RNA FnrS. Our results showed that a metE 5’ untranslated region with bp +30 to +68 deletion causes the metE gene expression decrease. Therefore, metE 5’ untranslated region is necessary for its gene expsresion, and this decrease is not affected by repressor MetJ. Howerer, the mechanism was different from the riboswitch of Bacillus clause. In addition, we also found that Hfq and FnrS are related to metE expression, and an v overexpression of FnrS inhibits the metE expression. The distortion of any secondary structure of fnrS or metE gene, has an effect on the expression of metE. Through our study, the regulation of Salmonella enterica metE gene were extensively studied and it would be very helpful for understanding of the regulation of other gene expression, which have a similar regulatory mechanism.